The in vivo examination of transcriptional control mechanisms in mammalian cells

Year: 1993

DOI: 10.7907/kt02-n508

In the investigations described in this thesis I have examined various
mechanisms involved in transcriptional control. The first chapter is a general
examination of the mechanisms used in transcriptional control. The second
chapter addresses issues associated with the selection of transcriptional start
site. This work shows that core promoter usage can be altered in a tissue specific
and environmentally-responsive manner. The third chapter of this
work describes a significant improvement in the technique of ligation mediated
PCR-aided in vivo footprinting and genomic sequencing. This
improvement in the quality of in vivo footprint data allows the pattern of
protein : DNA interactions to be obtained with greater signal to noise and for a
larger group of DNA sequences. The fourth chapter of this thesis uses these in
vivo footprinting techniques to investigate the mechanisms controlling the
transcription of the mouse interleukin-2 gene upon T cell stimulation. T cell
stimulation was shown to result in the coordinated occupancy of a number of
major groove binding proteins to the previously unoccupied IL-2 regulatory
region in vivo. Finally, the appendix describes in vivo footprints at the
differentiated-muscle-specific promoter of the delta-subunit of the nicotinic
acetylcholine receptor. Multiple protein : DNA interactions were seen at the
promoter both before and after muscle cell differentiation, suggesting that
transcriptional regulation of this gene occurs at the level of protein
replacement or an alteration of the ability of the assembled proteins to activate
transcription.